Microtubule dynamics during cell polarity and migration

描述(由申请人提供)：细胞骨架的极性对许多细胞行为是必不可少的，包括伤口愈合和趋化过程中的定向迁移。在平面极化的迁移细胞中，微管聚合动力学是时空调节的，是细胞定向迁移所必需的。这项建议的总体目标是确定微管在建立和维持上皮细胞平面极性和定向片状迁移过程中的作用机制，重点是CLAP末端家族的功能和动力学。TIPS是一组不同种类的蛋白质，由它们在细胞中生长的微管和末端的动态定位定义。在上皮片迁移过程中，TIPS是调节微管功能的主要候选者，因为它们可能为微管末端与皮质细胞骨架的调节相互作用提供接口。目前的研究集中在CLAPS上，因为它们与其他TIP的不同之处在于它们与迁移上皮细胞中的微管具有时空调节的联系。虽然卡环追踪细胞体中的微管和末端，但与其他尖端不同，卡环沿着迁移中的上皮细胞板层中的微管联系在一起。这种沿着板层微管的卡环的联系通过糖原合成酶激酶3？(GSK3？)，一个新出现的细胞极性途径的整合子。因为GSK3？被认为是在迁移细胞的前端局部失活的，据预测，细胞体中的CLAP-磷酸化降低了CLAP-微管的亲和力，而非磷酸化的CLAP与板层微管的结合稳定了这些微管。这一建议的具体假设是，CLAP介导的板层微管的稳定以及这些微管与皮质细胞骨架的相互作用是迁移上皮细胞持续平面极性所必需的。这一假设将通过生物化学方法和先进的活细胞显微镜在克隆的人类角质形成细胞中得到验证。在目标1中，将在细胞和体外分析依赖于GSK3的CLASP磷酸化，并将测试CLASP与微管或相关蛋白的相互作用是否受磷酸化的调节。在目标2中，将在平面极化、迁移细胞和体外用纯化的组分分析CLAP介导的微管聚合动力学的时空调节。它将被测试是否直接或间接调节微管动力学，这是否受GSK3的调节？磷酸化。在目标3中，通过RNA干扰将抑制迁移上皮细胞中的CLASP功能，并将通过使用活细胞旋转圆盘共聚焦显微镜分析板层突起、细胞-基质黏附和极性动力学来确定CLAP如何调控细胞的定向迁移。 公共卫生相关性：集体细胞迁移对于许多形态发生运动和损伤后角质形成细胞穿过基底膜的片状迁移是必不可少的。此外，细胞迁移的异常调节有助于肿瘤细胞的转移，而肿瘤细胞的集体迁移在肿瘤侵袭性中起着至关重要的作用。因此，了解平面上皮细胞极化和定向迁移的分子机制对于开发改善角质形成细胞或其他癌症的治疗方法以及创伤后皮肤再生至关重要。

DESCRIPTION (provided by applicant): Polarity of the cytoskeleton is essential for many cell behaviors, including directed migration during wound healing and chemotaxis. Microtubule polymerization dynamics are spatiotemporally regulated in planar polarized, migrating cells, and are required for cells to migrate directionally. The overall objective of this proposal is to determine the mechanisms by which microtubules function during establishment and maintenance of planar polarity and directed sheet migration of epithelial cells with an emphasis on the function and dynamics of the CLASP family of +TIPs. +TIPs are a heterogeneous group of proteins defined by their dynamic localization to growing microtubule plus ends in cells. +TIPs are prime candidates to mediate microtubule function during epithelial sheet migration because they may provide interfaces for regulated interactions of microtubule ends with the cortical cytoskeleton. The current study focuses on CLASPs because they are distinguished from other +TIPs by their spatiotemporally regulated association with microtubules in migrating epithelial cells. Although CLASPs track microtubule plus ends in the cell body, in contrast to other +TIPs CLASPs associate along microtubules in the lamella of migrating epithelial cells. This association of CLASPs along lamella microtubules is decreased through phosphorylation by glycogen synthase kinase 3? (GSK3?), an emerging integrator of cell polarity pathways. Because GSK3? is thought to be locally inactivated in the front of migrating cells, it is predicted that CLASP-phosphorylation in the cell body decreases CLASP-microtubule affinity, while association of non-phosphorylated CLASPs with lamella microtubules stabilizes these microtubules. The specific hypothesis of this proposal is that CLASP- mediated lamella microtubule stabilization and interactions of these microtubules with the cortical cytoskeleton are essential for persistent planar polarity of migrating epithelial cells. This hypothesis will be tested by biochemical methods and advanced live cell microscopy in clonal human keratinocyte cells. In Aim 1, GSK3?-dependent CLASP phosphorylation will be analyzed in cells and in vitro, and it will be tested whether CLASP interactions with microtubules or associated proteins are regulated by phosphorylation. In Aim 2, CLASP-mediated spatiotemporal regulation of microtubule polymerization dynamics will be analyzed in planar polarized, migrating cells, and in vitro with purified components. It will be tested whether CLASPs directly or indirectly regulate microtubule dynamics and whether this is regulated by GSK3? phosphorylation. In Aim 3, CLASP function will be inhibited in migrating epithelial cells by RNA interference, and it will be determined how CLASPs regulate directed cell migration by analyzing lamella protrusion, cell- matrix adhesion, and polarity dynamics using live cell spinning disk confocal microscopy. PUBLIC HEALTH RELEVANCE: Collective cell migration is essential for many morphogenetic movements and the sheet migration of keratinocytes across the basement membrane after injury. In addition, abnormal regulation of cell migration contributes to metastasis of tumor cells, and collective migration of tumor cells plays a crucial role in tumor invasiveness. Understanding the molecular mechanisms underlying planar epithelial cell polarity and directed migration is thus crucial to developing improved therapies for keratinocyte or other carcinomas, and for skin regeneration after wounding.